Epigenetic features in mammals include covalent modifications to histones, and methylation of cytosines. Their proper placement is fundamental to many biological processes. Assaying features in the epigenome is important for understanding human biology, diagnosing disease, monitoring responses to epigenome modifying drugs and facilitating development of new medicines. Such assays will also facilitate emerging therapeutics based on embryonic and induced pluripotent stem cells, which require modifying the cells to assume epigenetic states of differentiated cells. State-of-the-art assays for histone modifications use chromatin immunoprecipitation (ChIP), followed by genome wide sequencing. Methylated DNA can be identified by immunoprecipitation followed by sequencing, or by bisulfite sequencing. There are two fundamental limitations with all these approaches. First, they query only one epigenetic feature at a time. Epigenetic features arise in combinations, and those combinations rather than individual features regulate the underlying genes. Unless multiple features can be detected and measured simultaneously, it is not possible to know, with certainty, when combinations coexist on a given gene. Second, assays use populations of cells and report the average epigenetic states within the population, not the actual distribution of epigenetic states present on individual DNA molecules comprising the population. Third, ChIP often uses abundant amounts of chromatin making it impractical to assay multiple epigenetic features in rare or impossible to culture cells. In this application, we seek to develop a transforming technology, Single Chromatin molecule Analysis in Nanofluidics (SCAN) that can overcome each of these limitations and revolutionize epigenomic studies. In SCAN, chromatin molecules are bound to fluorescent probes recognizing distinct epigenetic features, then driven by voltage through nanofluidic channels where the fluorescent properties of single molecules are detected. By using multiple probes, each recognizing different features and carrying distinct fluorophores, we can directly detect their binding to individual molecules, allowing precise enumeration of multiple epigenetic features simultaneously. Our first-generation devices were operated in an analytical mode, simply counting features. Our second-generation devices were operated in a preparative mode, allowing us to sort and isolate molecules carrying defined epigenetic features. In this proposal, we seek to further develop this next generation epigenomics technology. First, we will modify the analytical device and analyte preparation to increase sample throughput by two orders of magnitude. Second, we will use the new analytical device to address selected questions in epigenomics. Third, we will use our preparative device to isolate chromatin with defined epigenetic features, sequence the DNA and compare our results to data obtained by current ChIP-seq methods.